We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.integral membrane proteins ͉ protein engineering ͉ protein folding

SUMMARY The flexibility of MAPK cascade responses enables regulation of a vast array of cell-fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems.

PEGylation is an attractive strategy to enhance the therapeutic efficacy of proteins with a short serum half-life. It can be used to extend the serum persistence and to reduce the immunogenicity of proteins. However, PEGylation can also lead to a decrease in the functional activity of the molecule to which it is applied. We constructed site-specifically PEGylated variants of anti-p185 HERϪ2 antibody fragments in the format of a monovalent single-chain variable fragment and a divalent miniantibody and characterized the antigen binding properties in detail. Mass-transport limited BIAcore measurements and binding assays on HER-2-overexpressing cells demonstrated that the immunoreactivity of the antibody fragments is fully maintained after PEGylation. Nevertheless, we found that the attachment of a 20-kDa polyethylene glycol (PEG) moiety led to a reduction in apparent affinity of approximately 5-fold, although in both formats, the attachment site was most distal to the antigen binding regions. This decrease in affinity was observed in kinetic BIAcore measurements as well as in equilibrium binding assays on whole cells. By analysis of the binding kinetics, we could pinpoint this reduction exclusively to slower apparent on rates. Through both experimental and computational analyses, we demonstrate that these reduced on-rates do not arise from diffusion limitations. We show that a mathematical model accounting for both intramolecular and intermolecular blocking mechanisms of the PEG moiety can robustly explain the observed binding kinetics. The results suggest that PEGylation can significantly alter the binding-competent fraction of both ligands and receptors and may help to explain some of the beneficial effects of PEGylation in vivo.

We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium. We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH. Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling.

Signaling networks that convert graded stimuli into binary, all-or-none cellular responses are critical in processes ranging from cell-cycle control to lineage commitment. To exhaustively enumerate topologies that exhibit this switch-like behavior, we simulated all possible two- and three-component networks on random parameter sets, and assessed the resulting response profiles for both steepness (ultrasensitivity) and extent of memory (bistability). Simulations were used to study purely enzymatic networks, purely transcriptional networks, and hybrid enzymatic/transcriptional networks, and the topologies in each class were rank ordered by parametric robustness (i.e., the percentage of applied parameter sets exhibiting ultrasensitivity or bistability). Results reveal that the distribution of network robustness is highly skewed, with the most robust topologies clustering into a small number of motifs. Hybrid networks are the most robust in generating ultrasensitivity (up to 28%) and bistability (up to 18%); strikingly, a purely transcriptional framework is the most fragile in generating either ultrasensitive (up to 3%) or bistable (up to 1%) responses. The disparity in robustness among the network classes is due in part to zero-order ultrasensitivity, an enzyme-specific phenomenon, which repeatedly emerges as a particularly robust mechanism for generating nonlinearity and can act as a building block for switch-like responses. We also highlight experimentally studied examples of topologies enabling switching behavior, in both native and synthetic systems, that rank highly in our simulations. This unbiased approach for identifying topologies capable of a given response may be useful in discovering new natural motifs and in designing robust synthetic gene networks.